TB40: Tris 20 mM pH = 8, 5 mM MgCl₂,10 mM DTT, 40 mM KCl, 20 µg/ml BSA.

Step 1:

Make 0.5 µM TDS-RNA9 hybrid:

- 2 µL 10 µM RNA9

- 1 µL 10 µM TDS

- 4 µL TB40X5

- 13 µL water

Use a PCR machine with the following program: 45˚C for 5 minutes, then reduce temperature by 1˚C every minute to 20˚C, then hold at 4˚C. 

Step 2: 

To 1 µL of 0.5 µM (0.5 pmol) hybrid, add 1 uL of Pol II 1.6 µM (1.6 pmol) (3.2-fold excess)

Incubate 10 minutes 24˚C.

Step 3:

Prepare NDS at 5 µM in TB40, add 1 µL (5 pmol) of that to the hybrid (10-fold excess).

Incubate at 37˚C for 15 minutes. 

At this point sample contains 3 µL at 0.167 µM.  

Step 4:  Stall and cleavage with BsaI (this is necessary for ligation to the beads via upstream handle)

Add 1 µL of 40 µM ATP/CTP/GTP in TB40, incubate 10 minutes RT

Add 1 µL of BsaI-HF (NEB) diluted X10 in TB40, incubate 15 minutes 37˚C.

At this point we have 5 µL of 100 nM stalled complex. However it should be assumed that true active stalled elongation complex constitutes at most 25% of this population (which gives an effective concentration of ~25 nM). The stalled elongation complex can be aliquotted in 0.5 µL aliquots, flash-frozen in liquid nitrogen, and kept at -80 degrees.